Ca2+ inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM
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Evaluation Summary:
Inactivation of ryanodine receptors (RyR1) is an important physiologic phenomenon disruption of which leads to skeletal muscle and heart diseases. By comparing cryoEM structures of RyR1 in closed, open, and inactivated states, this study provides structural insights into RyR1 calcium-dependent inactivation (CDI). The results rationalize how some disease-causing mutations in RyR1 eliminate CDI of the channel. The study will be of interest to ion channel structural biologists and physiologists studying skeletal muscle pathologies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Activation of the intracellular Ca 2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca 2+ surge, while elevated cytosolic Ca 2+ inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca 2+ conditions revealed an open and a closed-inactivated conformation. Ca 2+ binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca 2+ -inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca 2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2–S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca 2+ activation prerequisite for Ca 2+ inactivation and provides for a seamless transition from inactivated to closed conformations.
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Evaluation Summary:
Inactivation of ryanodine receptors (RyR1) is an important physiologic phenomenon disruption of which leads to skeletal muscle and heart diseases. By comparing cryoEM structures of RyR1 in closed, open, and inactivated states, this study provides structural insights into RyR1 calcium-dependent inactivation (CDI). The results rationalize how some disease-causing mutations in RyR1 eliminate CDI of the channel. The study will be of interest to ion channel structural biologists and physiologists studying skeletal muscle pathologies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The ryanodine receptor type 1 (RyR1) shows a biphasic response to Ca2+ - a Ca2+-dependent increase in activity (open probability) at low to moderate levels of Ca2+, and a Ca2+-dependent inactivation (CDI) at high Ca2+ concentrations. This study compares cryoEM structures of RyR1 embedded in nanodiscs in different states - closed, Ca2+-bound open, and Ca2+-bound closed (inactivated) - to gain insights into the structural correlates of RyR1 inactivation. The open and inactivated state structures are distinguished from previously published RyR1 structures by being obtained solely in the presence of physiological activators, Ca2+ and ATP, without other non-physiological activators (e.g. caffeine or PCB95) present.
Features revealed by the structures include that: Ca2+ remains bound in the high-affinity binding …
Reviewer #1 (Public Review):
The ryanodine receptor type 1 (RyR1) shows a biphasic response to Ca2+ - a Ca2+-dependent increase in activity (open probability) at low to moderate levels of Ca2+, and a Ca2+-dependent inactivation (CDI) at high Ca2+ concentrations. This study compares cryoEM structures of RyR1 embedded in nanodiscs in different states - closed, Ca2+-bound open, and Ca2+-bound closed (inactivated) - to gain insights into the structural correlates of RyR1 inactivation. The open and inactivated state structures are distinguished from previously published RyR1 structures by being obtained solely in the presence of physiological activators, Ca2+ and ATP, without other non-physiological activators (e.g. caffeine or PCB95) present.
Features revealed by the structures include that: Ca2+ remains bound in the high-affinity binding site in both open and inactivated state structures, although with important changes in binding interactions between the two states; two intersubunit salt bridges that form between EF hands and S2-S3 loop are important for the inactivated state; Ca2+ binds to the ATP binding pocket and there are changes in the interaction network of this pocket between open and inactivated states; lipids bind to a hydrophobic crevice in the transmembrane domain to stabilize the inactivated state.
Overall, the work provides nice structural insights into the Ca2+-dependent inactivation process in RyR1, an important physiological phenomenon. The results nicely complement and rationalize previously published functional studies that show disease-causing mutations in RyR1 that alter Ca2+-dependent inactivation.
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Reviewer #2 (Public Review):
The manuscript by Nayak et al. reported several cryo-EM structures of RyR1 with the aim to understand the inactivation mechanism of the channel. By comparing the structures from different functional states, they proposed a model how the rearrangement of RyR domains leads to a switch form the open to the inactivated state. The study is of great interest and the work is of fine quality.
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